SV40 tumor (T) antigen possesses four distinct antigenic determinants, sites I, II, III, and IV, recognized by SV40-specific H-2b-restricted cytotoxic T-lymphocytes (CTL) clones. SV40-transformed C57BL/6 (B6) mouse kidney cells, designated K-3, 1, 4, K-1, 4, and K-4, 1, have been isolated by immunological selection with SV40 T antigen site-specific CTL clones in vitro. The cells have lost the expression of all four antigenic sites and cannot be lysed by the CTL clones specific for antigenic sites I, II, III, and IV. To search for additional SV40-specific antigenic sites on SV40 T antigen, B6 mice were immunized with K-3,1,4 cells and stimulated spleen cells with K-3,1,4 cells in vitro. Repeated stimulation of the spleen cell culture with gamma-irradiated K-3,1,4 cells in the presence of IL-2 was necessary to generate CTL activity against K-3,1,4 cells. A new group of H-2Db-restricted CTL clones designated as Y-5 was isolated which were cytotoxic to K-3,1,4 cells. The antigenic site recognized by CTL clone Y-5, site V, was localized in the carboxy terminal half of the SV40 T antigen. By the use of a synthetic peptide corresponding to SV40 T antigen in the carboxy region, the antigenic site V was localized between amino acids 489 and 503.